In this R21 application, we aim to determine how the newly identified cellular restriction factors, known as interferon-inducible transmembrane (IFITM) proteins, modulate viral membrane fusion and entry, and in doing so, aid the development of novel antiviral therapeutics. The specific aims of this proposal are as follows. Aim 1: Test the hypothesis that IFITM proteins inhibit membrane fusion by preventing hemifusion. We will perform cell-cell and virion-cell fusion assays, including the use of single viral particle imaging technique, to determine the stages of the viral membrane fusion process blocked by IFITM proteins. Lipid analogs that modulate distinct stages of the membrane fusion process will be applied. We will explore the possibility that IFITM proteins restrict membrane fusion induced by cellular and developmental fusogens. Aim 2: Test the hypothesis that expression of IFITM proteins changes the lipid order of cell membranes and confers positive spontaneous curvature. We will perform fluorescence membrane labeling experiments to test the hypothesis that IFITM proteins cause the cell membrane to be more ordered, resulting in reduced fluidity and therefore less competency for fusion. We will also determine the effect of IFITM proteins on membrane curvature by reconstituting artificial liposomes with synthetic peptides corresponding to the functional domains in IFITM proteins. Cryo-EM tomography will be applied to directly visualize the curvature changes upon insertion of IFITM peptides into the artificial liposome vesicles. IFITM proteins are the first and so far only cellular restriction fators that are known to restrict viral membrane fusion and entry. Results from the proposed studies will provide novel insight as to how IFITM proteins restrict viral membrane fusion. These results will guide future comprehensive investigations into the biology of IFITM proteins, as well as their critical roles in restricting viral entry and infection.